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. Author manuscript; available in PMC: 2023 Sep 17.
Published in final edited form as: Science. 2023 Mar 16;379(6637):1140–1149. doi: 10.1126/science.abq5622

Figure 3. Dose dependent suppression of STMN2 cryptic splicing and polyadenylation by rASOs in iPSC-derived motor neurons with TDP-43 depletion.

Figure 3.

(A) Schematic representation of the exon 2a region of human STMN2 gene with TDP-43 binding sites and selected rASOs that show splice-modifying activity. (B) STMN2 mRNA restoration analyzed by qRT-PCR after treatment with 5 representative rASOs in iPSC-derived motor neurons depleted of TDP-43. Expression of TFRC mRNA was used as endogenous control. (C) qRT-PCR analysis of truncated STMN2 RNA levels after treatment with 5 representative rASOs in iPSC-derived motor neurons depleted of TDP-43. Expression of TFRC mRNA was used as endogenous control. (D) Immunoblot showing TDP-43 and stathmin-2 protein levels in iPS motor neurons treated with control or TDP-43 suppressing ASOs, subsequently treated with control or splice-rescuing rASOs 3, 4, or 5 to restore stathmin-2. Beta3 Tubulin used as an endogenous control. (E) Immunoblot showing TDP-43 and stathmin-2 levels in motor neurons depleted of TDP-43 with a targeted ASO and subsequently treated with control or rescue ASO-5. Linearity of antibody detection with 25% and 50% control treated motor neuron lysate loading controls included at the far-left side of the blot. Immunoblot quantified in (F). Each lane and data point represents an independently differentiated and ASO treated neuronal culture. Error bars are SEM. Statistical significance was determined by 1-way ANOVA with Dunnett correction. ****, p <0.0001; ***, p < 0.001; **, p < 0.01; *, p <0.05.